Protein kinases (PKs) are enzymes which catalyze the phosphorylation of specific serine, threonine, or tyrosine residues in cellular proteins. These post-translational modifications of substrate proteins act as a molecular switch, playing a central role in diverse biological processes such as the control of cell growth, metabolism, tumor microenvironment (such as VEGFR), differentiation, and apoptosis. Aberrant, excessive, or, more generally, inappropriate PK activity has been observed in several disease states including malignant proliferative disorders such as RET gain of function mutations for medullary thyroid cancer (MTC) and other human malignancies, ITD (Internal Tandem Duplication)-mutations in Flt3 for acute myelogenous leukemia (AML), c-Kit mutation for gastrointestinal stromal tumors (GIST), and Bcr-abl rearrangement for chronic myelogenous leukemia (CML). In addition, the activation and/or overexpression of tyrosine kinases such as Trk-A, Trk-B, Trk-C, and RET has been linked to severe pain in cancer patients, especially pancreatic cancer. Many tyrosine kinases are homologous to each other; the inhibition of one tyrosine kinase will also likely produce some inhibitory activity on other tyrosine kinases. For example, imatinib has been used as a therapeutic not only for CML patients (based on the inhibition of Bcr-abl kinase), but also GIST cancer patients (based on the inhibition of c-Kit kinase). The recent advance of basic and clinical studies on the tyrosine kinases has demonstrated that many tyrosine kinases may be targeted by drugs. For example, more than a dozen new drugs targeting VEGFR2, Bcr-abl, Flt3, platelet-derived growth factor receptor (PDGFR), and c-Kit have been approved in the last decade. A few targets and their implications for cancer therapy are briefly described.
RET
In 1985, the RET (Re-arranged during transfection) gene was identified as a novel oncogene activated through DNA rearrangement (Takahashi, M. Cell, 1985, 42, 581-588).
MTC (Medullary Thyroid Carcinoma) is a malignancy of the C cells in the thyroid gland. MTC can be either sporadic or familial as part of Multiple Endocrine Neoplasia type 2 syndromes MEN2A and MEN2B. Both familial (about 95%) and sporadic (about 50%) MTC display gain-of-function point mutations in the RET proto-oncogene leading to increased pro-survival signaling and cell growth. RET signaling sustains tumorigenesis. As a result, blocking this RET signaling provides an “Achilles' heel” treatment avenue for MTC patients. Multiple endocrine neoplasia 2B is an inherited syndrome caused primarily by the M918T mutation in the kinase domain of the RET receptor, while multiple endocrine neoplasia 2A is primarily caused by mutation of C634 (Santoro, M. et al Science, 1995, 267, 381). Neither mutation is located in the ATP binding site. Isolated familial MTC is caused by different mutations either in RET extracellular or intracellular domains, including the mutations V804M and V804L, which target the gatekeeper residue in the ATP binding site of the kinase. Conventional systemic treatment for MTC has been generally poorly effective, with 56% of patients experiencing post-surgical relapses due to early metastasis (Wells, Jr. S. A. et al, Clin. Cancer Res. 2009, 15, 7119-7123).
PTC (Papillary Thyroid Carcinoma) arises from follicular thyroid cells. In PTC, RET is targeted by chromosomal rearrangements that result in the in-frame fusion of part of its intracellular domain with the 5′-end of heterologous genes. About 60,000 new cases of thyroid cancer were estimated in the United States in 2012. Distant metastases are observed at presentation in fewer than 5% of the differentiated thyroid cancer patients, and recurrent disease occurs in 10-15%. Treatment of recurrent disease mainly includes surgery and radioiodine. However, treatment of radioactive refractory PTC disease is still an unmet medical need (Schlumberger, M. Thyroid, 2009, 19, 1393-1400).
Lung cancer is the leading cause of cancer-related mortality. Treatment paradigms for non-small-cell lung cancer (NSCLC), which accounts for the majority of all lung cancers, have shifted away from a histologic diagnosis to a diagnosis utilizing molecular subtypes. Some “molecular driver mutations” can result in constitutively active mutant signaling proteins, such as EGFR and ALK. Recently, some studies identified RET kinase fusions (KIF5B-RET and more rarely RET/PTC variants) in about 1% of patients with adenocarcinoma-type NSCLC. As rapidly as four years after the identification of ALK fusions in lung cancer, one ALK inhibitor for NSCLC was approved by FDA. Therefore, RET-positive NSCLC patients may similarly benefit from specific targeted therapies (Hutchinson, K. E. Nat. Med. 2012, 18, 349-351).
CMML is a myeloproliferative disorder that presents monocytosis. Myeloproliferative neoplasms are frequently associated with aberrant constitutive tyrosine kinase activity resulting from chimeric fusion genes or point mutations. Two novel fusion genes in CMML, BCR-RET and FGFR10P-RET, have been reported. Blocking activity of RET in CMML is important for reestablishing signaling homeostasis, regenerating proper hematopoietic differentiation, and controlling aberrant tumorigenic signaling (Ballerini, P. et al, Leukemia 2012, 1-6).
RET overexpression in a subset of ER-positive breast cancers was also recently identified. In situ hybridization (a technique to determine the presence or absence of genetic sequences in tissue) of a cohort of 245 invasive breast cancers detected RET and GFRα1 mRNA in 29.7% and 59.4% of the tumors, respectively. The majority of these tumors were ER-positive. A similar finding was reported in a survey of breast cancer cell lines. Furthermore, qPCR analysis of a small panel of breast tumor primary cells detected preferential expression of RET mRNA in ER-positive samples. Finally, these studies are strengthened by microarray studies showing that in a set of 36 breast cancer samples, RET expression positively correlated with ER expression. The role of RET in tamoxifen-resistant breast cancer was further validated in the cell-based assay format by Dr. Isacke and her colleagues. In ERα-positive breast cancer cells, activation of the receptor tyrosine kinase RET by its ligand GDNF resulted in increased ERα phosphorylation on Ser118 and Ser167, and estrogen-independent activation of ERα transcriptional activity. In vitro RET downregulation resulted in a 6.2-fold increase in sensitivity of MCF7 cells to the antiproliferative effects of tamoxifen, whereas GDNF stimulation had a protective effect against the drug. In the tamoxifen-resistant MCF7 cells, targeting RET restored tamoxifen sensitivity. Finally, examination of two independent tissue microarrays of primary human breast cancers revealed that expression of RET protein was significantly associated with ERα-positive tumors and that there was a two-fold increase in the number of RET-positive tumors in patients who subsequently developed invasive recurrence after adjuvant tamoxifen treatment (Morandi, A. Trends in Mol. Med. 2011, 17, 149-157).
VEGFR
Vascular endothelial growth factor (VEGF) is an important signaling protein involved in both vasculogenesis and angiogenesis. As its name implies, VEGF activity is restricted mainly to cells of the vascular endothelium, although it does have effects on a limited number of other cell types. In vitro, VEGF has been shown to stimulate endothelial cell mitogenesis and cell migration. VEGF also enhances microvascular permeability and is sometimes referred to as vascular permeability factor. VEGFR kinases have been used as a target for solid tumors, such as malignancies that are highly vascular like renal carcinoma, glioblastoma, and liver cancers (Bhargava, P. Curr Oncol Rep, 2011, 103-111).
FLT3
While cure rates for acute myeloid leukemia (AML) have improved over the past four decades, survival remains suboptimal. Five-year survival for patients under 60 years old is only 40%. The standard of care for most newly diagnosed patients with AML consists of induction chemotherapy with infusional cytarabine and an anthracycline.
Mutations in the FMS-like tyrosine kinase 3 (FLT3) gene characterize more than 30% of AML cases. FLT3 internal tandem duplication (ITD) mutations, accounting for approximately 23% of AML cases, are associated with a particularly poor prognosis. The prognostic implications of FLT3/D835 point mutations found at diagnosis, comprising approximately 7% of cases, are not yet well established. Inhibiting FLT3 and its mutation would be advantageous.
c-KIT
c-Kit is a receptor tyrosine kinase that normally controls the function of primitive hematopoietic cells, melanocytes and germ cells. It has become clear that uncontrolled activity of c-Kit contributes to formation of an array of human tumors. The unregulated activity of c-Kit may be due to overexpression, autocrine loops, or mutational activation. This makes c-Kit an excellent target for cancer therapies in these tumors, especially GIST and AML (Von Mehren, M. Clin. Colorectal Cancer, 2006, S30-40).
TRK
Tropomyosin-related kinases (Trks) are receptor tyrosine kinases normally expressed in neuronal tissue where they play important role in both development and function. The Trk receptor family is composed of three members (A, B, and C) activated by specific ligands called neurotrophins. Each Trk receptor contains an extracellular domain, a transmembrane region, and an intracellular domain which upon binding of their respective ligand (nerve growth factor (NGF) for TrkA, brain-derived growth factor (BDNF) and NT-4/5 for TrkB, and NT3 for TrkC), triggers oligomerization of the receptors, phosphorylation of specific tyrosine residues in the kinase domain, and down-stream signal transduction pathways, including survival, proliferation, and differentiation in normal and neoplastic neuronal cells. Deregulation of TrkA and TrkB and their cognate ligands has been described in numerous types of cancers including prostate, breast, colorectal, ovarian, lung, pancreas, melanoma, thyroid, and neuroblastoma, and occurs mainly through wild type receptor overexpression, activation, amplification, and/or mutation. Importantly, increased Trks activation in tumor tissues correlates with an aggressive phenotype and poor clinical outcome. Additionally, Trks, as well as RET, play a role for the perineural invasion and associated cancer pain.
PDGFR
Platelet-derived growth factor acts as a potent mitogen and chemotactic factor for various cells such as fibroblasts, smooth muscle cells, mesenchymal cells, and brain glial cells. Abnormal PDGF-induced cell proliferation has been proposed to lead to proliferative disorders. There is a need for a PDGFR inhibitor that offers therapeutic benefits for proliferative disorders such as gastrointestinal stromal tumors (GIST), glioma, and melanoma.